Hydroxylating activity of tyrosinase and its dependence on hydrogen peroxide

The aim of this work was to study the hydroxylation of N, N-dimethyltyramine (DMTA) by tyrosinase in the presence of hydrogen peroxide, a reaction that does not take place without the addition of the hydrogen peroxide. Some properties of this hydroxylating activity are analyzed. The kinetic parameters of mushroom tyrosinase toward hydrogen peroxide (K(m) = 0.5 mM, V(m) = 11 microM/min, V(m)/K(m) = 2.2 x 10(-2) min(-1)) and toward DMTA (K(m) = 0.3 mM, V(m) = 4.8 microM/min, V(m)/K(m) = 16 x 10(-2) min(-1)) were evaluated. There was a lag period, which was similar to the characteristic lag of monophenolase activity at the expense of molecular oxygen. The length of this lag phase decreased with increasing hydrogen peroxide concentration, and disappeared at approximately 0.5 mM H(2)O(2). However, the lag was longer with higher DMTA concentrations. The pH optimum range for this hydroxylating activity was 6.0 to 7.0. The lag also varied with pH, increasing at pH values higher than 6.7. The presence of hydrogen peroxide is necessary for the oxidation of DMTA, as is the presence of active enzyme since the reaction was completely inhibited when selective tyrosinase inhibitors were added.